An aim of known light microscopes and microscopy methods is to determine the height of a specimen surface. The specimen surface can be understood on the one hand to be the outer boundary of an object to be examined. However, it can also be understood to be the surface of an inner part of the object to be examined, for example biological cells or cell components in an aqueous solution. Such measurements are relevant in particular for characterising technical surfaces and for deriving roughness measurement values and topographies.
The aforementioned height determination is possible with a generic microscopy method for examining a microscopic specimen. It is provided in this method that illuminating light is emitted with a light source device to the specimen, that specimen light coming from the specimen is guided with optical imaging means to a detector unit, that the specimen light is measured with the detector unit to produce a plurality of specimen recordings, that height information for a respective plurality of lateral regions of the specimen is obtained from each specimen recording, wherein the height information of each specimen recording is limited to one height measurement range in each case and the height measurement ranges of different specimen recordings differ from each other, and that an overall image is calculated from the specimen recordings, in which overall image height information of the different specimen recordings is brought together.
A generic light microscope for examining a microscopic specimen comprises a light source device to emit illuminating light to the specimen, optical imaging means to guide illuminating light to the specimen and to guide specimen light coming from the specimen, a detector unit to measure the specimen light to produce a plurality of specimen recordings, electronic control and evaluation means which are designed to obtain from each specimen recording height information for each of a plurality of lateral regions of the specimen, wherein the height information of each specimen recording is limited to one height measurement range in each case and the height measurement ranges of different specimen recordings differ from each other, and wherein the electronic control and evaluation means are additionally designed to calculate an overall image from the specimen recordings, in which overall image height information of the different specimen recordings is brought together.
Bringing together the height information of different specimen recordings can be understood in that the height information of one specimen recording is no longer provided independently of the height information of further respective specimen recordings, but instead the height information of each of the specimen recordings is expressed relative to a common reference point. Height information of one specimen recording can thereby be meaningfully compared with height information from another specimen recording.
Height examinations with confocal microscopes are known for example. In this case, the specimen is scanned with illuminating light which is initially focussed onto a certain height plane. Meanwhile, a specimen image is recorded as the aforementioned specimen recording. This specimen image can for example be evaluated to ascertain whether a specimen region, that is to say a certain lateral region of the specimen, lies exactly in the illuminated height plane or is at a distance from it. The specimen is then moved with an adjustable specimen table in the height direction, that is to say: in the direction of the optical axis extending from the specimen to an objective of the light microscope. A second specimen image is then recorded and evaluated. The relationship between the height of a lateral region lying in the case of the first specimen image in the illuminated height plane and the height of another lateral region located in the case of the second specimen image in the illuminated height plane is now to be determined. For this purpose, the adjusting height is detected, by which the specimen is displaced in height between the recordings of the two images. Height information of two specimen recordings is then brought together through the knowledge of the adjusting height. A disadvantage here is that cost-intensive adjusting elements are required to determine this height with great precision.
In order to simultaneously examine a plurality of lateral regions, the spinning disc method with a Nipkow disc can be used in confocal microscopy. In this case, a disc is used which has a plurality of holes, through which a plurality of lateral regions are illuminated. By rotating the disc, a scan is carried out in a lateral direction. For specimen recordings with different height measurement ranges, a displacement of the specimen in the height direction is also required here and this must conventionally be determined via a high-precision actuator system.
In addition, vibrations or impacts can arise here, through which a height position of the specimen varies during the measurements. In most cases, expensive vibration-damping tables are therefore used.
The same problems arise in microscopy with structured illumination, wherein specimen images are usually produced for different lattice images and are taken into calculation to produce a high-resolution image.
Also in the case of a separate simultaneous recording of confocal and non-confocal light portions, a height displacement of the specimen must be carried out, whereby this is associated with high costs for precise adjusting units and undesirable vibration effects.
Furthermore, the chromatic confocal principle is known for obtaining height information. In this case, an optical element with chromatical effect is used, of which the refractive power is dependent upon wavelength. In this way, light can be focussed, in dependence upon its wavelength, onto different height planes. A broadband light source or a tunable light source with sequential confocal detection can be used. In dependence upon the intensity of light of different wavelengths, the height of a lateral region of the specimen can be concluded. A height measurement range is defined through the different focussing of light of different wavelengths. If the specimen has greatly differing heights which go outside of this height measurement range, the specimen must also be moved in the height direction in this measurement method. This displacement distance must conventionally also be detected with precise adjusting means. In addition, vibrations of the specimen table, for example, have great disadvantageous effects upon the measurement result.
The same problems also exist in the case of nano-profilometry methods. Here, a specimen is positioned so that it is located in the ascending or descending flank of the axial response function of a wide-field confocal microscope. If the relative reflectivities of the different specimen lateral regions are known through a previous calibration step, the height profile can be directly determined. However, the height measurement range is limited, meaning that the specimen must often be displaced in the height direction and a further specimen recording is necessary. In order to link the height information of these different recordings, accurate knowledge of the height adjustment of the specimen is required.
There are numerous further measurement methods but which are respectively limited to one height measurement range and require a displacement of the specimen relative to the illumination and detection optical path. All these conventional methods have the disadvantages that a high-precision relative displacement of the specimen with cost-intensive adjusting elements is necessary and that the measurement results can nonetheless be greatly influenced by vibrations.